Lecture Series 8LThe Eucaryotic Genome and

It E iIts Expression

Reading AssignmentsReading Assignments

R d Ch 8 R d Ch 8 •• Read Chapter 8 Read Chapter 8 Control of Gene ExpressionControl of Gene Expression

•• Skim Chapter 9Skim Chapter 9How Genes and Genomes EvolveHow Genes and Genomes EvolveHow Genes and Genomes EvolveHow Genes and Genomes Evolve

A The Eucaryotic GenomeA. The Eucaryotic Genome

Al h h h DNA i Al h h h DNA i •• Although eucaryotes have more DNA in Although eucaryotes have more DNA in their genomes than bacteria and archaea, their genomes than bacteria and archaea, in some cases there is NO apparent in some cases there is NO apparent in some cases there is NO apparent in some cases there is NO apparent relationship between genome size and relationship between genome size and organism complexity.organism complexity.organism complexity.organism complexity.

Amoeba dubia is the big winner at 670 Billionbase pairs per cell and an uncertain phylogeny!

A The Eucaryotic GenomeA. The Eucaryotic Genome

U lik b i l h l DNA U lik b i l h l DNA •• Unlike bacterial or archaeal DNA, Unlike bacterial or archaeal DNA, eukaryotic DNA is separated from the eukaryotic DNA is separated from the cytoplasm by being contained within a cytoplasm by being contained within a cytoplasm by being contained within a cytoplasm by being contained within a nucleus. nucleus. Th i iti l mRNA t s i t f th DNA Th i iti l mRNA t s i t f th DNA •• The initial mRNA transcript of the DNA The initial mRNA transcript of the DNA gets modified before it is exported to gets modified before it is exported to the cytoplasmthe cytoplasmthe cytoplasm.the cytoplasm.

A The Eucaryotic GenomeA. The Eucaryotic Genome

Th f h i lTh f h i l ll d b ddi ll d b ddi •• The genome of the singleThe genome of the single--celled budding celled budding yeast contains genes for the same yeast contains genes for the same metabolic machinery as bacteria as well metabolic machinery as bacteria as well metabolic machinery as bacteria, as well metabolic machinery as bacteria, as well as genes for protein targeting in the cell.as genes for protein targeting in the cell.

A The Eucaryotic GenomeA. The Eucaryotic Genome

Th f h l i ll l Th f h l i ll l •• The genome of the multicellular The genome of the multicellular roundworm roundworm Caenorhabditis elegansCaenorhabditis eleganscontains genes required for intercellular contains genes required for intercellular contains genes required for intercellular contains genes required for intercellular interactions.interactions.Th m f th f it flTh m f th f it fl h s f h s f •• The genome of the fruit flyThe genome of the fruit fly has fewer has fewer genes than that of the roundworm. Many genes than that of the roundworm. Many of its sequences are homologs of of its sequences are homologs of of its sequences are homologs of of its sequences are homologs of sequences on roundworm and mammalian sequences on roundworm and mammalian genes.genes.gg

Chromatin in a developing salamander ovum

Levels of chromatin packing

Chromatin

Chromatin, detail

B Mutations: Heritable B. Mutations: Heritable Changes in Genes•• Mutations in DNA are often expressed as Mutations in DNA are often expressed as

abnormal proteins. However, the result abnormal proteins. However, the result ppmay not be easily observable phenotypic may not be easily observable phenotypic changes.changes.

•• Raw materials for evolution to operate.Raw materials for evolution to operate.•• Some mutations appear only under certain Some mutations appear only under certain Some mutations appear only under certain Some mutations appear only under certain

conditions, such as exposure to a certain conditions, such as exposure to a certain environmental agent or condition.environmental agent or condition.gg

B Mutations: Heritable B. Mutations: Heritable Changes in Genes•• Point mutations (silent, missense, Point mutations (silent, missense,

nonsense, or framenonsense, or frame--shift) result from shift) result from ))alterations in single base pairs of DNA.alterations in single base pairs of DNA.

Categories and consequences of point mutations: Base-pair substitution

Categories and consequences of point mutations: Base-pair indels

The molecular basis of sickle-cell disease: a point mutation

B Mutations: Heritable B. Mutations: Heritable Changes in Genes•• Chromosomal mutations (deletions, Chromosomal mutations (deletions,

duplications, inversions, or translocations) duplications, inversions, or translocations) p )p )involve large regions of a chromosome. involve large regions of a chromosome.

Alterations of chromosome structure

C Repetitive SequencesC. Repetitive Sequences

Hi hl i i DNA i i Hi hl i i DNA i i •• Highly repetitive DNA is present in up to Highly repetitive DNA is present in up to millions of copies of short sequences. It is millions of copies of short sequences. It is not transcribed Its role is unknownnot transcribed Its role is unknownnot transcribed. Its role is unknown.not transcribed. Its role is unknown.

•• Rem: Some moderately repetitive DNA Rem: Some moderately repetitive DNA sequences, such as telomeric DNA is sequences, such as telomeric DNA is f d h d f hf d h d f hfound at the ends of chromosomes.found at the ends of chromosomes.

C Repetitive SequencesC. Repetitive Sequences

S d l i i DNA S d l i i DNA •• Some moderately repetitive DNA Some moderately repetitive DNA sequences, such as those coding for sequences, such as those coding for ribosomal RNA’s are transcribedribosomal RNA’s are transcribedribosomal RNA s, are transcribed.ribosomal RNA s, are transcribed.

•• Up to three rRNAs result, two go to the Up to three rRNAs result, two go to the large subunit and one goes to the small large subunit and one goes to the small

b ib isubunit.subunit.

Moderately repetitive DNA sequencesModerately repetitive DNA sequences

Part of a family of identical genes for ribosomal RNA

C Repetitive SequencesC. Repetitive Sequences

S d l i i DNA S d l i i DNA •• Some moderately repetitive DNA Some moderately repetitive DNA sequences are transposable, or able to sequences are transposable, or able to move about the genome These are known move about the genome These are known move about the genome. These are known move about the genome. These are known as Transposons.as Transposons.T s s s j m f m l t l T s s s j m f m l t l •• Transposons can jump from place to place Transposons can jump from place to place on the chromosome by actually moving or on the chromosome by actually moving or by making a new copy inserted at a new by making a new copy inserted at a new by making a new copy, inserted at a new by making a new copy, inserted at a new location.location.

Transposons in corn

Insertion sequences, the simplest transposons

Insertion of a transposon and creation of direct repeats

Anatomy of a composite transposon

Types of DNA sequences in the human genomeExons (regions of genes codingfor protein, rRNA, tRNA) (1.5%)

RepetitiveDNA thatincludestransposable

Introns andregulatoryp

elementsand relatedsequences(44%)

g ysequences(24%)

UniquenoncodingDNA (15%)

RepetitiveDNAunrelated totransposableelements(about 15%)

Alu elements(10%)

Simple sequenceDNA (3%)

Large-segmentduplications (5–6%)

D The Structures of D. The Structures of Protein-Coding Genes•• A typical proteinA typical protein--coding gene has coding gene has

noncoding internal sequences (introns) as noncoding internal sequences (introns) as noncoding internal sequences (introns) as noncoding internal sequences (introns) as well as flanking sequences that are well as flanking sequences that are involved in the machinery of transcription involved in the machinery of transcription involved in the machinery of transcription involved in the machinery of transcription and translation in addition to its exons or and translation in addition to its exons or coding regions.coding regions.g gg g

•• These are usually single copy genes.These are usually single copy genes.

D The Structures of D. The Structures of Protein-Coding Genes•• Some Some eucaryotic eucaryotic genes form families of genes form families of

related genes that have similar sequences related genes that have similar sequences related genes that have similar sequences related genes that have similar sequences and code for similar proteins. These and code for similar proteins. These related proteins may be made at different related proteins may be made at different times and in different tissues. times and in different tissues.

•• Some sequences in gene families are Some sequences in gene families are q gq gpseudogenes, which code for nonfunctional pseudogenes, which code for nonfunctional mRNA’s or proteins.mRNA’s or proteins.

Gene Families

Pseudogenes

D The Structures of D. The Structures of Protein-Coding Genes•• Differential expression of different Differential expression of different

genes in the genes in the --globin family ensures globin family ensures genes in the genes in the --globin family ensures globin family ensures important physiological changes during important physiological changes during human development.human development.pp

The evolution of human -globin and -globin gene families

E Differential Gene ExpressionE. Differential Gene Expression

E i E i i b i b •• Eucaryotic Eucaryotic gene expression can be gene expression can be controlled at the transcriptional, controlled at the transcriptional, posttranscriptional translational and posttranscriptional translational and posttranscriptional, translational, and posttranscriptional, translational, and posttranslational levels.posttranslational levels.

Eucaryotes: Eucaryotes one promoter per gene

Genes can be expressed with d ff ffdifferent efficiencies

The cell controls its metabolism by controlling the The cell controls its metabolism by controlling the level level and activity of enzymes that participate in and activity of enzymes that participate in each each reaction or reaction reaction or reaction seriesserieseach each reaction or reaction reaction or reaction series.series.

E. Differential Gene Expressionp

?

Genes aren’t lost during development, but rather each cell becomes more and more restricted in its fate, expressing ultimately a specific subset of genes responsible for defining its specific function.

E Differential Gene ExpressionE. Differential Gene Expression

• We can show that all the information to make an organism resides in every cell resides in every cell.

• Theoretically, every cell could be used to regenerate a regenerate a genetically identical adult (clone).

• Cells that are capable • Cells that are capable of regenrating a fully formed adult are called totipotent.p

Why not synthesis all the genes y y gall the time at a moderate level?

• Too expensive• Levels need to be Levels need to be

controlled• Some products are m p

incompatible• Need change in g

response to signals• Development

Principles of gene controlPrinciples of gene control

Constitutive expression• Constitutive expression– A gene is expressed at

approximately the same approximately the same levels all the time: (for example: a housekeeping example: a housekeeping gene)

• Regulated expressionRegulated expression– Gene expression in

response to a signalresponse to a signal

F Transcriptional ControlF. Transcriptional Control

Th j h d f l f Th j h d f l f i i •• The major method of control of The major method of control of eucaryotic eucaryotic gene expression is selective transcription, gene expression is selective transcription, which results from specific proteins binding which results from specific proteins binding which results from specific proteins binding which results from specific proteins binding to regulatory regions on DNA.to regulatory regions on DNA.

RNA polymerases:RNA polymerasesthe more the merrier…

F Transcriptional ControlF. Transcriptional Control

A i f “ l” i i f A i f “ l” i i f •• A series of “general” transcription factors A series of “general” transcription factors must bind to the promoter before RNA must bind to the promoter before RNA polymerase can bind polymerase can bind polymerase can bind. polymerase can bind.

•• Whether RNA polymerase will initiate Whether RNA polymerase will initiate t s i ti ls d ds th bi di t s i ti ls d ds th bi di transcription also depends on the binding transcription also depends on the binding of regulatory proteins, activator proteins, of regulatory proteins, activator proteins, and repressor proteinsand repressor proteinsand repressor proteins.and repressor proteins.

RNA pol II requires many “general” transcription factors

Phosphorylation of RNA pol II allows RNA processing proteins to ride on its tail

Action of distal enhancers and transcription activators

Repressors/Silencers too!

Distal control elements can also be silencerssilencersbe silencerssilencers

• Enhancers and silencers bind specialized transcription Enhanc rs an s nc rs n sp c a z transcr pt on factors that can promote or interfere with the formation of a functional transcription initiation complex

F Transcriptional ControlF. Transcriptional Control

Th DNATh DNA bi di d i f DNAbi di d i f DNA•• The DNAThe DNA--binding domains of most DNAbinding domains of most DNA--binding proteins have one of four binding proteins have one of four structural motifs: helixstructural motifs: helix turnturn helix zinc helix zinc structural motifs: helixstructural motifs: helix--turnturn--helix, zinc helix, zinc finger, leucine zipper, or homeodomain.finger, leucine zipper, or homeodomain.

Three of the major types of DNA-binding domains in transcription factors

Homeodomains

L in ZippZinc Finger

Leucine Zipper

F Transcriptional ControlF. Transcriptional Control

A l i f hi il A l i f hi il •• Acetylation of histone tails promotes Acetylation of histone tails promotes loose chromatin structure that permits loose chromatin structure that permits transcription to more readily occurtranscription to more readily occurtranscription to more readily occur.transcription to more readily occur.

A simple model of histone tails and the effect of histone acetylation

Chromatin changes

Transcription

RNA processing

mRNA degradation

Translation

Protein processingand degradation

HistoneHistonetails

DNADNAdouble helix Amino acids

availablefor chemicalmodification

(a) Histone tails protrude outward from a nucleosome( ) p f

(b) Acetylation of histone tails promotes loose chromatinstructure that permits transcription

Unacetylated histones Acetylated histones

Combinatorial control regulation concept

G. Posttranscriptional ControlG. Posttranscriptional Control

B B i i h l h l •• Because Because eucaryotic eucaryotic genes have several genes have several exons, alterative mRNAs can be generated exons, alterative mRNAs can be generated from the same RNA transcriptfrom the same RNA transcriptfrom the same RNA transcript.from the same RNA transcript.

•• This alternate splicing can be used to This alternate splicing can be used to d diff t t i sd diff t t i sproduce different proteins.produce different proteins.

•• The stability of mRNA in the cytoplasm The stability of mRNA in the cytoplasm b l d b h bi di f b l d b h bi di f can be regulated by the binding of can be regulated by the binding of

proteins.proteins.

Alternative RNA splicing

Chromatin changes

Transcription

RNA processing

mRNAdegradation

Translation

Protein processingand degradation

Exons

DNA

PrimaryPrimaryRNAtranscript

RNA splicing or

mRNA

Alternative RNA splicingAlternative RNA splicing

G Posttranslational ControlG. Posttranslational Control

•• Proteasomes degrade proteins targeted Proteasomes degrade proteins targeted for breakdown.for breakdown.for breakdown.for breakdown.

Degradation of a protein by a proteasome

Proteasomes

H Regulation of Gene H. Regulation of Gene Expression in Bacteria•• An operon consists of a promoter, an An operon consists of a promoter, an

operator, and structural genes. Promoters operator, and structural genes. Promoters operator, and structural genes. Promoters operator, and structural genes. Promoters and operators do not code for proteins, and operators do not code for proteins, but serve as binding sites for regulatory but serve as binding sites for regulatory proteins. proteins.

•• When a repressor protein binds to the When a repressor protein binds to the p pp poperator, transcription of the structural operator, transcription of the structural genes is inhibited.genes is inhibited.

Repressor Bound to an Operator Blocks Transcription

Minor Groove Major Groove

H Regulation of Gene H. Regulation of Gene Expression in Bacteria•• The expression of The expression of bacterial genes bacterial genes is is

regulated by: inducible operatorregulated by: inducible operator––repressor repressor regulated by inducible operatorregulated by inducible operator repressor repressor systems, repressible operatorsystems, repressible operator––repressor repressor systems (e.g., both negative control), and systems (e.g., both negative control), and systems that increase the efficiency of a systems that increase the efficiency of a promoter (e.g., positive control).promoter (e.g., positive control).

•• Repressor proteins are coded by Repressor proteins are coded by constitutive regulatory genes.constitutive regulatory genes.

The tryptophan operon: a biosynthetic operon controlled by a repressorrepressora biosynthetic operon controlled by a repressorrepressor

NOTE! The operator and promoter sequences overlapoverlapp q pp

This gene has its own promoter and is constitutively yexpressed at a low levels.

Allostery! The repressor protein is activated (to repress!) by binding tryptophan

The trp operon: regulated synthesis of repressible enzymes

The trp operon: regulated synthesis of repressible enzymes

The lac operon:a catabolic operon controlled by a repressorrepressor

This gene has its own promoter and is constitutively expressed at a low levels.

This is an inducibleinducible operon

The lac operon: regulated synthesis of inducible enzymes

The lac operon: regulated synthesis of inducible enzymes

H Regulation of Gene H. Regulation of Gene Expression in Bacteria•• The efficiency of RNA polymerase can be The efficiency of RNA polymerase can be

increased by regulation of the level of increased by regulation of the level of increased by regulation of the level of increased by regulation of the level of cyclic AMP, which binds to CAP (cAMP cyclic AMP, which binds to CAP (cAMP activator protein). activator protein).

•• The CAPThe CAP––cAMP complex then binds to a cAMP complex then binds to a site near the promoter of a target gene, site near the promoter of a target gene, p g g ,p g g ,enhancing the binding of RNA polymerase enhancing the binding of RNA polymerase and hence transcription.and hence transcription.

The lac operon: positive controlThe lac operon positive control

The presence of glucose prevents the transcription of the lac operon.

Positive control: cAMP activator protein

CAP

CAPCAP

CAP

Positive control: cAMP activator protein

CAP

CAP

Operons: ReviewOperons: Review

This is a repressiblerepressible operon

This is a repressiblerepressible operon

I Comparison of ControlI. Comparison of ControlFeatures in Bacteria & EEucaryaB h l l d l B h l l d l •• Bacteria have multiple genes under single Bacteria have multiple genes under single control: operonscontrol: operons

•• Eucarya have multiple RNA polymerasesEucarya have multiple RNA polymerases•• Simple vs. Complex Transcription FactorsSimple vs. Complex Transcription Factorsp p pp p p•• Local vs. Distal Control: Enhancers/SilencersLocal vs. Distal Control: Enhancers/Silencers

E t t d ith Ch tiE t t d ith Ch ti•• Eucarya must contend with ChromatinEucarya must contend with Chromatin

What are eucaryotic-specific What are eucaryotic specific control issues?

• Distal control elements• Chromatin

Transcriptional control• Chromatin• Splicing

Post-transcriptional • mRNA transport• Protein transport

Post transcriptional control

Protein transport• Protein modifications

l ll l

Post-translational control

• Multicellularity Signal transduction